The objective of this proposal is to analyze the kinetic properties and elucidate the regulatory mechanisms of molecular interactions of the T cell receptor (TCR) and coreceptor CD8 with peptide-major histocompatibility complex (pMHC) molecules, which are the first step of antigen recognition by the T cell and trigger adaptive immune responses. Our studies differ from previous binding studies in fluid phase using soluble molecules (three-dimensional or 3D binding) by analyzing these interactions in situ at the two- dimensional (2D) cross-junctional interface between a T cell and a surrogate antigen presenting cell using assays invented by the PI, which keeps the TCR and CD8 in their native environment with all regulatory mechanisms intact. Our preliminary data show drastic differences between 2D and 3D binding, including 1) 2D TCR/pMHC kinetics correlates with T cell response; 2) 2D analysis enables in situ measurements of TCR/pMHC/CD8 trimeric interactions and of antigen-induced TCR-CD8 cooperation via Lck-dependent signaling; 3) 2D parameters can be perturbed by pharmacological agents that inhibit signaling and/or disrupt the lateral organization of the cel membrane and cytoskeleton. Our hypothesis is that 2D kinetics of TCR and CD8 for pMHC binding determines a variety of functions of T cells in different stages, including discrimination f pathogens from self-antigens, thymic selection during development, and activation. This broad hypothesis will be tested in three integrated specific aims: 1) elucidate the mechanisms underlying how and why 2D kinetic parameters determine T cell responsiveness by determining whether and how they can be related to 3D kinetic parameters; 2) compare the 2D kinetics of pMHC binding to naive vs. activated, effector, and memory T cells, the resulting calcium signaling, and their correlations with T-cell responses; and 3) analyze 2D kinetics of pMHC binding to thymocytes in different maturation states and the resulting calcium signaling to determine thresholds for thymic selection. These studies will provide a new paradigm for understanding the kinetic requirements for interaction and cooperation of the TCR and CD8 as related to signaling initiation upon pMHC binding and serve as a foundation for a better understanding of a variety of immune functions.